Indole Derivatives as New Structural Class of Potent and Antiproliferative Inhibitors of Monocarboxylate Transporter 1 (MCT1; SLC16A1)

The solute carrier (SLC) monocarboxylate transporter 1 (MCT1; SLC16A1) represents a promising target for the treatment of cancer; however, the MCT1 modulator landscape is underexplored with only roughly 100 reported compounds. To expand the knowledge about MCT1 modulation, we synthesized a library of 16 indole-based molecules and subjected these to a comprehensive biological assessment platform. All compounds showed functional inhibitory activities against MCT1 at low nanomolar concentrations and great antiproliferative activities against the MCT1-expressing cancer cell lines A-549 and MCF-7, while the compounds were selective over MCT4 (SLC16A4). Lead compound 24 demonstrated a greater potency than the reference compound, and molecular docking revealed strong binding affinities to MCT1. Compound 24 led to cancer cell cycle arrest as well as apoptosis, and it showed to sensitize these cancer cells toward an antineoplastic agent. Strikingly, compound 24 had also significant inhibitory power against the multidrug transporter ABCB1 and showed to reverse ABCB1-mediated multidrug resistance (MDR).


■ INTRODUCTION
Cancer is a leading cause of death in over 70% of the countries worldwide and accounts for 30% of all premature deaths of all noncommunicable diseases. 1 Chemotherapy is the most important noninvasive treatment available. However, firstand even second-line anticancer drugs become often ineffective 2 due to the occurrence of multidrug resistance (MDR) of cancer cells, by either mutations/changed drug targets 3 or reduced drug uptake/upregulation of efflux transporters, 2,4,5 amongst other reasons. Although tremendous advances have been made within the last two decades by the discovery of novel anticancer drug targets, 4,6−8 the frequent occurrence of MDR forces research to focus on new pharmacological targets for the exploration of novel cancer drug targets of the future.
Cancer cells go through a process of metabolic reprogramming to contest changes in the metabolism such as hypoxia due to poor vascularization or increased glucose consumption to support enhanced cell growth. 9 The tumor microenvironment and nutrition status have been acknowledged as driving factors in tumor growth, prognosis, and survival rate. 10,11 Particularly, cancer cells within solid tumors are prone to hypoxia, leading to an overexpression of hypoxia-inducible factor-1 (HIF-1), triggering glucose transporter 1 (GLUT1) expression to increase glucose uptake. 9,11,12 This changes the basic energy supply from mitochondrial oxidative phosphor-ylation (as in normoxic cells) to glycolytic metabolism. 9,11,13 Thus, hypoxic cells convert glucose to pyruvate and lactate, and abundant lactate is extruded into the tumor microenvironment by monocarboxylate transporter 4 (MCT4). Strikingly, this lactate becomes imported by MCT1 and converted into pyruvate feeding the tricarboxylic acid (TCA) cycle 9,13 and, thus, aerobic energy production. MCT1 overexpression is found in many cancer cells, 10,13−16 and its support of cancer migration, invasion, and metastasis 13 has been recognized. Hence, MCT1 represents a promising drug target for the development of novel anticancer agents.
To explore indoles as potential novel molecular−structural class of MCT1 inhibitors, we extended the structural diversity of indoles by synthesizing 16 derivatives including their comprehensive in vitro assessment against MCT1-mediated transport, cancer cell viability, and cancer cell cycle, but also against selected drug transporters. We were able to elucidate important structure−activity relationships (SARs) and discovered potent and antiproliferative agents that may represent the lead molecules for further clinical evaluations.   (26,28) led to a strong increase of inhibitory power. Particularly, compound 24 with its 4-methoxy substitution showed the highest MCT1 transport inhibition with an IC 50   Figure 3 provides the concentration-effect curves of the most potent MCT1 inhibitors of the indole series, 24 (A) and 30 (B), compared to the reference MCT1 inhibitor 8. Inhibitory Power against MCT4. MCT4 is a key protein in the metabolic reprogramming of hypoxic tumor cells. Thus, we investigated the MCT1 inhibitors for their potential activity against MCT4, applying a pH-sensitive fluorescence assay as described in the literature 35,40 with minor modifications. In short, functional MCT4 inhibition prevents the extrusion of acidic lactate, which acidifies the cytosol of the MCT4expressing cancer cell line MDA-MB-231. 22,41,42 The degree of inhibition is reflected in the magnitude of pH reduction. However, none of the 16 evaluated indole derivatives showed a pH reduction in MCT4-expressing MDA-MB-231 22,41,42 cells (data not shown).
Antiproliferative Activity against MCT1-Expressing Cancer Cell Lines. MCT1-expressing cells have an extraordinary energy demand and rely on pyruvate metabolism, through either lactate conversion or pyruvate consumption. Inhibition of MCT1 blocks lactate and pyruvate influx into cancer cells, which prevents their processing in the TCA cycle. Over time, the viability of cells exposed to an MCT1 inhibitor is impaired due to pyruvate deprivation, which is compensated by upregulating glucose metabolism�leading to an overconsumption of glucose. To confirm the antiproliferative nature of the indole derivatives, we investigated the compounds toward the MCT1-expressing cell lines A-549 14,15 and MCF-7. 16 Cancer cell viability was assessed via a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as reported earlier. 43 As can be seen from Table 2, the entire compound class of indoles bears considerable cytotoxicity toward MCT1-expressing cancer cell lines, being more toxic than the reference MCT1 inhibitor 8. Interestingly, the 2-pyridyl derivatives 29− 31 including the second most potent lead MCT1 inhibitor 30 had the greatest effect on cancer cell viability. Figure 4 shows In the next step, the cell toxicity 43 of the compounds was analyzed against the non-MCT1-expressing, noncancerous murine embryonic fibroblast cell line NIH/3T3 to analyze whether the observed effects relate to malignant MCT1-(over)expression. Strikingly, none of the indole derivatives had a considerable impact on the cell viability of NIH/3T3 cells up to a concentration of 50 μM ( Figure 5). This finding led us to the conclusion that the observed effects are due to the selective inhibition of MCT1, which is expressed in A-549 14,15 and MCF-7 16 cancer cell lines.   To prove that the cell death of MCT1-expressing cancer cells results from glucose deprivation, we conducted additional MTT-based cell viability experiments with compounds 24 and 30 supplementing glucose (25 and 50% of the original glucose concentration of 4.5 g/L every 12 h) over a time span of 72 h. Strikingly, we could, for the first time, prove that the supplementation of glucose prevents cell toxicity of the MCT1 inhibitors 24 ( Figure 6A) and 30 ( Figure 6B), thus confirming that glucose deprivation is the main cause of cell death mediated by MCT1 inhibitors.    cases, the compounds sensitized the cells against 33 by factors of 2.5 and 2.6, respectively, as indicated by the shift of the concentration-effect curve to the left, demonstrating their capability to increase susceptibility toward antineoplastic agents. Table 3 provides the GI 50 values of 33 either without or with increasing concentrations of either compound 24 or 30.
Cell Cycle Distribution Analysis of Compound 24. To prove that indole derivatives impair cancer cell proliferation and to confirm their antiproliferative nature, we conducted a cell cycle distribution analysis via a propidium iodide (PI) flow cytometry assay, as reported earlier. 44−46 A-549 cells were exposed to the most potent MCT1 inhibitor, 24 (5 μM), and the distribution of the cells in the cell cycle phases sub-G 1 , G 0 / G 1 , S, and G 2 /M was calculated compared to untreated A-549 cells. The antineoplastic agent 33 (5 μM) was used as a reference. Figure 8 shows the histograms for untreated (A), 24treated (B), and 33-treated cells (C). Compound 24 caused a disruption of the cell cycle of A-549 cancer cells, indicated by a shift from the predominant G 0 /G 1 phase ( Figure 8A) to the apoptotic sub-G 1 phase as well as the G 2 /M phase ( Figure 8B). A similar G 2 /M arrest could be observed for reference compound 33 ( Figure 8C); however, MCT1 inhibitor 24 led to a larger fraction of cells into the sub-G 1 phase, indicating induced apoptosis. This experiment discovered that compound 24 inhibits cell proliferation, causing cell cycle arrest and, eventually, apoptosis. To confirm our findings, we conducted an apoptosis assay using compound 24.
Apoptosis Assay. As compound 24 increased the sub-G 1 cell cycle fraction of A-549 cells, we investigated its capability to induce apoptosis in a PI/Annexin V-FITC staining assay, as reported earlier. 44,46−51 In short, cells that were stained with PI only were considered necrotic cells (Q1), while cells stained with both PI and Annexin V were late apoptotic (Q2), and cells stained with Annexin V only were early apoptotic (Q4). Cells negative on both PI and Annexin V were considered living cells (Q3). Compound 24 (5 μM) increased the percentage of total apoptotic cells by a factor of 13 from 0.51 to 6.68% when compared to the untreated A-549 cells, indicating that 24 exhibited cytotoxicity against the A-549 cancer cell line via inducing apoptosis. Figure 9 shows the flow cytometry scatter plot of untreated (A) compared to that of 24-treated A-549 cells (B).
Inhibitory Activity against Multidrug Transporters ABCB1, ABCC1, and ABCG2. Multidrug transporters of the ATPbinding cassette (ABC) superfamily play a major role in MDR and the distribution of antineoplastic agents. 2,4,5 To provide a detailed picture of the pharmacological profile of the indole derivatives, we investigated their modulatory nature on the most prominent ABC transporters ABCB1 (P-glycoprotein, Pgp), ABCC1, (multidrug resistance-associated protein 1, MRP1), and ABCG2 (breast cancer resistance protein, BCRP). Here, we conducted calcein AM (ABCB1), 2,52−59   daunorubicin (ABCC1), 52,58−60 and pheophorbide A (ABCG2) 2,52−60 assays using ABCB1-expressing A2780/ ADR, ABCC1-expressing H69AR, and ABCG2-expressing MDCK II BCRP cells, respectively. Figure 10 shows the screening results of the compounds at a concentration of 10 μM against ABCB1 (A), ABCC1 (B), and ABCG2 (C), and Table 4      or cell toxicity of the compounds. As this effect of partial inhibition frequently occurs in calcein AM assays, 57,58,63,66 we verified our results in terms of the IC 50 values in a daunorubicin assay, as reported earlier. 52,60 Strikingly, the lead compounds 24 ( Figure 11C) and 30 ( Figure 11D) had inhibitory potencies of 1.49 and 2.77 μM, respectively, in the daunorubicin assay while having a similar I max compared to reference ABCB1 inhibitor 34.
Compounds 22−23, on the other hand, showed inhibitory activities against all three evaluated ABC transporters (IC 50 + SEM ≥ 20%) and, thus, qualify as the so-called "focused pan-ABC transporter inhibitors". 71 Multitarget agents with these polypharmacological profiles have been of increasing interest within recent years, 52−54,60,72−74 and the herein discovered compounds 22−23 build a good starting point for the development of novel multitarget agents stretching from ABC to SLC transporters.
Efficacy of Compounds 24 and 30 against ABCB1-Expressing Cells. The surprisingly high inhibitory power against ABCB1 prompted us to investigate whether the compounds (1.0, 2.0, 3.5, and 5.0 μM) were capable of sensitizing ABCB1-expressing A2780/ADR cells toward the antineoplastic agents 33. Thus, we applied an MTT-based efficacy assay as reported earlier. 2,55−59 Figure 12 shows the concentration-effect curves of 33 without and with the supplementation of compounds 24 (A) or 30 (B). Both compounds (5 μM) completely sensitized ABCB1-expressing A2780/ADR cells against compound 33. This sensitization is indicated by a shift of the concentration-effect curve of compound 33 from the right (=high concentrations of 33 needed to induce A2780/ADR cell death) to the left (=lower concentrations of 33 needed to induce A2780/ADR cell death). The concentration-effect curve of compound 33 in combination with 5 μM of either compound 24 or 30 resembles the concentration-effect curve of the sensitive cell line A2780. The half-maximal reversal concentration (EC 50 ) of the compounds was 0.793 ± 0.039 μM (24; Figure 12C) and 1.22 ± 0.01 μM (30; Figure 12D), which matched the concentration range of their IC 50 values. Thus, their potentiating effect with respect to the antiproliferative agent 33 against ABCB1-expressing A2780/ADR cells is due to the inhibition of ABCB1. Therefore, compounds 24 and 30 were dually effective against MCT1-and ABCB1-expressing cells (Table 5).
Computational Analyses. Molecular Docking of Lead MCT1 Inhibitors. To get further insights into the ligand− transporter interactions and the structural background of MCT1 inhibition, molecular docking of the reference MCT1 inhibitors 7 and 8, as well as the lead MCT1 inhibitors 24 and 30, was applied using a recently released cryo-EM structure of human MCT1 (PDB ID: 7CKR). 19 Figure 13 shows the human MCT1 cryo-EM structure with compound 7 embedded within the transmembrane regions.
The most potent lead MCT1 inhibitor 24 had a very low free binding energy of −9.5 kcal·mol −1 , which is comparable to the free binding energy of reference inhibitor 7. Additionally, 24 formed three conventional hydrogen bonds with Lys38, Ser154, and Arg313 along with one carbon−hydrogen bond with Ser371. It additionally developed two electrostatic π− cation interactions with Lys38 and hydrophobic interactions (π−sigma, π−cation−alkyl) with Pro37, Leu66, Val282, and Pro406. The second most potent MCT1 inhibitor, 30, had with −9.7 kcal·mol −1 a similar free binding energy compared to lead MCT1 inhibitor 24, which is significantly lower compared to reference inhibitor 8 and comparable to reference inhibitor 7. The interaction profile of compound 30 parallels the one of    Figure 14 outlines the interaction profiles of compounds 7 (B), 8 (C), 24 (D), and 30 (E).
The found results revealed that the lead MCT1 inhibitors 24 and 30 had binding affinities similar to the golden reference compound 7. Compounds 7, 8, 24, and 30 targeted the same binding pocket that was already earlier identified by a structure-focused study. 19 Furthermore, the identified interactions of compounds 7, 8, 24, and 30 resembled in the same binding pocket embedded by not only Lys38 and Arg313 (shared amongst all four compounds) but also Pro37, Leu66, and Pro406 (shared amongst compounds 7, 24, and 30) despite the structural variation between reference inhibitors and indole derivatives. Thus, these interactions found the very basis of MCT1 inhibition.
Determination of Physicochemical Properties. Molecular−structural characteristics, such as hydrogen-(H)-bond donors, H-bond acceptors, or rotatable bonds are important for the pharmacokinetics and pharmacodynamics of drugs. 60,72 Also, certain physicochemical properties, such as the calculated octanol/water partition coefficient (CLog P), molecular weight (MW), molar refractivity (MR), or the topological polar surface area (TPSA), shape cellular penetration and drug

Journal of Medicinal Chemistry
pubs.acs.org/jmc Article effectiveness. 60,72 Thus, these and other important parameters of the investigated compounds were calculated by applying the online web service SwissADME (http://www.swissadme. ch). 75 Table 6 provides these data including the overall drug likeliness of the compounds according to the Lipinski rule-offive and their interaction profile with the pharmacokineticinfluencing ABC transporters ABCB1, ABCC1, and ABCG2.

■ DISCUSSION AND CONCLUSIONS
Standing of Indole Derivatives. The discovery of novel drug targets opening up new therapeutic options is the challenge of medicinal chemistry today. However, the discovery is only the first of multiple steps to access these discovered targets for pharmacotherapy. MCT1 has been described as a critical factor of cancer survival for several years now, 10,13−16 and yet, the fund of compounds addressing this SLC transporter with sufficient efficacy and preferable pharmacological profile is highly limited. 18−31 Only roughly 100 compounds have been described to address this underexplored drug target, 18−31 including the drugs and druglike compounds that were the first ligands with poor inhibitory activity, such as 1 and 3. 31 Potent MCT1 inhibitors with affinities in the single-digit nanomolar concentration range or even at subnanomolar concentrations indeed exist; 18,28,29,32,34 however, these compounds exclusively belong to the few known molecular−structural classes, as outlined in the Introduction section, and do not contribute to structural diversity. Up to date, only compound 4 reached the clinical drug development stage (NCT01791595, completed 2022); 18 the screening of chemical space as well as the exploration and exploitation of novel molecular−structural compound classes necessitates. Indole represented a promising scaffold considering the initial indole-and indole-like scaffold-containing hit molecules addressing MCT1. 13,35−37 In our study, we provided important SARs of indole derivatives and, thus, explored these as a novel molecular− structural class of MCT1 inhibitors. We obtained compounds with higher inhibitory potency compared to a significant fraction of the ∼100 reported MCT1 inhibitors in the literature, 18−31 including pteridine derivatives, 33 most coumarin derivatives, 25 29 or 7 34 as well as many cinnamic acid derivatives. 22,24 However, three aspects have to be considered to put the bioactivity profile of indole derivatives into the right perspective: (i) As MCT1 inhibitors are barely known with only roughly 100 representatives in the literature, 18 34 The within this work used reference inhibitor 8 presented a similar picture in the literature from submillimolar to low micromolar concentration ranges. 21,22,25 Thus again, the overall comparability of the literature data is limited.
(iii) To oppose the limited data situation and reduce data variation, the development of novel, easier-to-apply, reliable, and robust assays is an important factor, also in terms of the exploration of novel pharmacological drug targets. Particularly, the functional 3-BP cell viability assay that was used within our work is such a new assay that has not found its way into broad experimental use yet. 20,39 This lack of application is of course accompanied by uncertainty with respect to the outcome data. However, our assay has several very pronounced advantages compared to assay setups as reported in point (i): (a) most assays conducted with respect to MCT1 are based on radioactivity measurement 21,22,24,25,29,31,33,34 and thus are constrained to regulatory requirements, which limit general application. 3-BP, on the other hand, is a regular chemical without regulatory constraints, promoting general use; (b) radiotracer assays are accompanied�apart from regulatory constraints�by complex protocols, which makes the determination of full-blown concentration-effect curves or even high-throughput screenings a challenge. In contrast, the 3-BP assay is an easy-to-perform assay with a protocol comparable to other functional and/or MTT-based assays as described for other protein (super)families, as, for example, ABC transporters; 2,7,[52][53][54][55][56][57][58][59][60][61]63,66,72 and (c) furthermore, the 3-BP assay demonstrated extreme reliability and robustness, as can be seen from the low deviations depicted either in Table  1 as well as in Figure 3.
Targeting Monocarboxylate Transport as Potential Anticancer Strategy. Lead compound 24 decreased the cell viability of MCT1-expressing cancer cells due to pyruvate and subsequent glucose deprivation and increased the susceptibility of MCT1-expressing cells to the antineoplastic agent 33. Further biological investigations showed that compound 24 induced cell cycle arrest and cancer cell apoptosis. These findings present compound 24 as one of the most effective MCT1 inhibitors with an optimized additional pharmacological profile, demonstrating its aptitude as an adjuvant therapeutic together with first-line antineoplastic agents in use to treat several types of cancers.
None of the 16 evaluated compounds demonstrated an inhibitory feature against MCT4, the other promising anticancer target. However, the indole-like compound 10 was shown previously to inhibit the mitochondrial pyruvate carrier (MCP1). 37 Thus, an interaction of the herein presented indole derivatives is thinkable, which would be of advantage as MCP1 has been identified as another potential anticancer drug target. 78,79 Further investigations are warranted to explore these novel potential therapeutic options.
Polypharmacology of Indole Derivatives. Interestingly, indoles turned out to have a rich polypharmacological profile as we identified many inhibitory interactions with ABC transporters. MDR caused by the upregulation of ABC transporters remains until today another big obstacle in anticancer therapy, 2,4,5 e.g., by conferring resistance of cancer cells against antineoplastic agents, such as compound 33. Compound 24 inhibited the multidrug transporter ABCB1 with an IC 50 of roughly 1 μM, and it completely sensitized ABCB1-expressing cancer cells at 5 μM, which is a moderate potency in terms of the efficacy against ABCB1. In essence, in addition to its already optimized pharmacological profile, compound 24's polypharmacological nature addressing two phylogenetically unrelated but functionally similar membranebound transporters not only makes it the first synthesis-derived member of its kind but also represents a very promising starting structure for ongoing biological investigations.
In line with the polypharmacological nature of compound 24 is the discovery of the focused 71 pan-ABC transporter inhibitors 22 and 23, which additionally addressed MCT1, presenting themselves as the first synthesis-derived pan-ABC/ SLC transporter modulators. The usefulness of pan-agents has been broadly discussed in the literature, 52−54,60,72−74 and particularly intersuperfamily addressing molecules may be of great use to further explore target space and identify as well as validate potential pharmacological drug targets of the future. ■ EXPERIMENTAL SECTION Chemistry. Materials. Chemicals and solvents were purchased from Omkar Traders (Mumbai, India), Sigma-Aldrich (Mumbai, India), and Sisco Research Laboratories (Mumbai, India) and were used without further purification. All reactions were carried out under an inert atmosphere, and thin-layer chromatography (TLC) was applied to monitor the reaction progress using an aluminum plate coated with silica gel 60 F 254 (Merck Millipore, Billerica, MA). Chloroform/methane (95%/5%) was used as an eluent, and the results were viewed under a UV cabinet (Desaga, Biostep, Burkhardtsdorf, Germany) at a wavelength of 254 nm. Both column chromatography on silica gel (60−120 μm; Merck, Mumbai, India) and flash chromatography (Combiflash RF, Teledyne ISCO, NE, Lincoln) were used to accomplish the chromatographic purification using dichloromethane/methane 98%/2%.
The identity of compounds 17−32 was determined by Fourier transform infrared (FTIR, Spectrum RXI, Perkin Elmer Spectra, Waltham, MA) and 1 H NMR spectroscopy (Bruker Advance DX 400 MHz, Billerica, MA). The chemical shifts (δ) were expressed in ppm in relation to the internal standard tetramethylsilane, and multiplicity of signals was indicated as singlet (s), doublet (d), doublet of doublets (dd), triplet (t), triplet of doublets (td), pentet (p), and multiplet (m). The molecular mass of compounds was determined by liquid chromatography-mass spectrometry (LCMS) analysis (LCMS-8040, Shimadzu, Kyoto, Japan), and all compounds are >95% pure by highperformance liquid chromatography (HPLC, Shimadzu, Kyoto, Japan). The melting point of the compounds was determined using the melting point apparatus (Veego, Mumbai, India).
1H-Indole 3-carbaldehyde (13). Intermediate 13 was prepared as described in the literature with minor modifications. 80 After being cooled in an ice−salt bath, N,N-dimethylformamide was subjected to phosphoryl chloride (POCl 3 ; 2 equiv). The resulting reaction mixture was stirred for 20 min before a solution of indole (12, 1 equiv) in N,N-dimethylformamide (25 mL) was added. The mixture was stirred for 4 h at room temperature. After adding a trace quantity of crushed ice, the mixture was basified with 5 M sodium hydroxide (pH = 14). Intermediate 13 precipitated after stirring for 1 h at room Journal of Medicinal Chemistry pubs.acs.org/jmc Article temperature, which was filtered, dried, and used without further purification and characterization. Ethyl 2-(3-Formyl-1H-indol-1-yl) (14). Cesium carbonate (Cs 2 CO 3 ; 1.5 equiv) was added to a solution of intermediate 13 (1 equiv) in DMF (5 mL), and the reaction mixture was stirred for 10 min at room temperature. Ethyl bromoacetate (1.1 equiv) was added, and the reaction mixture was stirred for 6 h at room temperature. Icecold water was added to the mixture, forming intermediate 14.
The precipitate was filtered, dried, recrystallized from ethanol, and used without further characterization.
2-(3-Formyl-1H-indol-1-yl) Acetic Acid (15). NaOH (4 equiv) was added to a stirred solution of intermediate 14 in ethanol at 0°C, followed by further stirring for 1 h. The solvent was concentrated, and eventually, the reaction mixture was acidified with 1 M hydrochloric acid. The precipitated intermediate 15 was filtered off, dried, recrystallized from ethanol, and used without further characterization. (16). Intermediate 15 (1 equiv) and methyl cyanoacetate (1 equiv) in methanol (10 mL) were mixed with catalytic amounts of piperidine, and the mixture was stirred for 7 h. The solvent was evaporated under reduced pressure, and the mixture was acidified with 2 M HCl forming intermediate 16. The formed precipitate was filtered off, dried, recrystallized from ethanol, and used without further characterization.

2-(3-(2-Cyano-3-methoxy-3-oxoprop-1-en-1-yl)-1H-indol-1-yl) Acetic Acid
General Procedure for the Preparation of Compounds (17−16). POCl 3 (1.2 equiv) was added to a stirred solution of intermediate 16 (1 equiv) in dichloromethane and catalytic amounts of pyridine, which was stirred for 20 min at 0°C. The respective substituted aromatic amine (1.1 equiv) was added at 0°C, and the mixture was stirred for 6 h, followed by stirring for 30 min at room temperature. The reaction mixture was poured into ice-cold water (100 mL), and the respective target compound was extracted with EtOAc (2 × 100 mL). The combined organic phases were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure yielding the respective target compound.
Inhibitory Activity against MCT1. A functional 3-BP cytotoxicity assay was conducted as described in the literature 20 Inhibitory Activity against MCT4. Determination of intracellular pH was carried out using the pH-sensitive dye 2′,7′-bis-(2carboxyethyl)-5/6-carboxyfluorescein acetoxymethyl ester (BCECF-AM; Sigma-Aldrich, St. Louis, MO). Briefly, 90 μL of an MDA-MB-231 cell suspension (50,000 cells/well) was added to a black flatbottom 96-well microplate (SPL Life Science, Gyeonggi-do, Republic of Korea). The plate was incubated overnight at 37°C under a 5% CO 2 -humidified atmosphere. Then, 10 μL of the test compounds (10 μM) dissolved in DPBS (Genetix Biotech Asia, New Delhi, India) was added. The plates were further incubated for 12 h at 37°C under a 5% CO 2 -humidified atmosphere. After incubation, the supernatant was removed and 100 μL of a BCECF-AM (5 μM) solution was added to each well, and the plates were further incubated for 1 h at 37°C under a 5% CO 2 -humidified atmosphere. Afterward, the dye loading solution was removed and the cells were washed two times with water for injection (Genetix Biotech Asia, New Delhi, India), and subsequently, the intracellular pH was determined. The pH calibration was carried out for each plate by adding 100 μL of intercellular pH calibration buffers for pH 4.5, 5.5, 6.5, and 7.5 (Invitrogen by Thermo Fisher Scientific, Eugene, OR) with 10 μM of nigericin and 10 μM of valinomycin to each well, followed by an incubation period of 30 min at room temperature. Finally, the fluorescence was spectrophotometrically determined (excitation: 430/ 90 nm; emission: 535 nm) using a SpectraMax iD3 multi-mode microplate reader (Molecular Devices, San Jose, CA). For intracellular pH determination, the fluorescence ratio between the emission wavelengths 430 nM and 490 nm was determined and plotted against the calibration curve.
Antiproliferative Activity against MCT1-Expressing Cancer Cell Lines. To determine the growth inhibitory potential of compounds 17−32, 20 μM of each compound either at concentrations between 1 and 100 μM (A-549 or MCF-7) or at 10 and 50 μM (NIH/3T3) was pipetted into colorless flat-bottom 96-well microplates. Thereafter, 180 μL of a cell suspension (5000 cells/well) of either A-549, MCF-7, or NIH/3T3 cells was added and subsequently kept for 72 h at 37°C under a 5% CO 2 -humidified atmosphere. The cell viability as well as the GI 50 values were determined as described above. 43 The glucose deprivation analyses were conducted by adding 4.5 μL of glucose (final concentrations 25% and 50% of the original 4.5 g/L) to each well at an interval of 12 h over a period of 72 h to the 180 μL cell suspension of A-549 cells in a colorless flat-bottom 96-well microplate (SPL Life Sciences, Gyeonggi-do, Republic of Korea). Cell viability was determined as described above.
Efficacy of Compounds 24 and 30 against MCT1-Expressing Cells. The ability of the most potent MCT1 inhibitors 24 and 30 to enhance the susceptibility of the MCT1-expressing cell lines A-549 14,15 toward antineoplastic agent 33 was investigated. For this purpose, 10 μL of compounds 24 or 30 (50 nM, 100 nM, or 500 nM) was transferred onto a colorless flat-bottom 96-well microplate and complemented with 180 μL of a cell suspension containing either approximately 5,000 A-549 cells. A dilution series of compound 33 at concentrations between 50 nM and 50 μM was generated, and 10 μL of each concentration was transferred onto the plate. The observed effects of compounds 24 and 30 were compared to the effect of compound 33 alone on A-549. The plates were incubated for 72 h at 37°C and a 5% CO 2 -humidified atmosphere, before subsequent processing with the regular MTT assay as described above. 43 Cell Cycle Distribution Analysis of Compound 24. A standard PI staining procedure was used as described earlier 44−46 to determine the effect of compounds 24 and 33 on cell cycle progression, followed by flow cytometry analysis (FACS Aria SORP and FACS Aria Fusion, BD Biosciences, Franklin Lakes, NJ). In brief, A-549 cells were seeded onto a 6-well plate (250,000 cells/well) and incubated for 24 h at 37°C under a 5% CO 2 -humidified atmosphere before being observed under a microscope. The cells were treated with compound 24 (5 μM) or 33 (5 μM) and further incubated for 24 h at 37°C under a 5% CO 2 -humidified atmosphere. Then, the cells were harvested and washed twice with cold PBS (Genetix Biotech Asia, New Delhi, India) before being fixed overnight in ice-cold 70% (v/v) ethanol at 4°C. The cells were again washed two times with PBS and subsequently resuspended with RNase (100 μg/mL; HiMedia, Mumbai, India) and their DNA was stained with PI (40 μg/mL; Sigma-Aldrich, Mumbai, India) before being incubated in the dark for 10 min. The DNA content was measured at the Indian Institute of Technology Bombay (IITB, Mumbai, India) by applying flow cytometry.
Apoptosis Assay. A PI/Annexin V-FITC double staining method was used to carry out a flow cytometry-based apoptosis assay, as reported earlier. 44,46−51 For this purpose, a PI/Annexin V-FITC apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ) was used following the manufacturer's instructions. A-549 cells were seeded onto 6-well plates with a density of 500,000 cells/well and incubated for 24 h at 37°C under a 5% CO 2 -humidified atmosphere. Subsequently, the cells were exposed to compound 24 (5 μM) followed by a further incubation period of 24 h at 37°C under a 5% CO 2 -humidified atmosphere. The cells were trypsinized, washed with PBS, and resuspended in 1x binding buffer (1,000,000 cells/mL). One hundred microliters of test solution containing 100,000 cells treated with a mixture of PI (5 μL) and Annexin V-FTIC (5 μL) was incubated in the dark for 10−15 min at room temperature. Four hundred microliters of binding buffer was added, and the cells were resuspended again just before the flow cytometry analysis by using flow cytometry.
Compounds 17−32 were pipetted in a volume of 20 μL at a concentration of 100 μM onto a clear 96-well flat-bottom plate (Brand, Wertheim, Germany), adding 160 μL of cell suspension containing either 30,000 cells/well (calcein AM) or 45,000 cells/well (daunorubicin and pheophorbide A) in either phenol red-free RPMI-1640 (A2780/ADR and H69AR) or phenol red-free DMEM (MDCK II BCRP) without further supplements. Compounds 17−32 were incubated with the cells for 30 min before adding the respective fluorescence dye [20 μL of calcein AM (3.125 μM), daunorubicin (30 μM), or pheophorbide A (5 μM); final concentrations: calcein AM: 0.3125 μM; daunorubicin: 3 μM; pheophorbide A: 0.5 μM] to each well. The fluorescence increase was measured over a time period of 30 min in 30 s intervals in the case of calcein AM (excitation: 485 nm; emission: 520 nm) using a Paradigm microplate reader (Beckman Coulter, Brea, CA), while the average fluorescence value per well was measured after an incubation period of 180 min and 120 min in the case of daunorubicin and pheophorbide A, respectively (excitation: 488 nm; emission: 695/50 nm), using an Attune NxT flow cytometer (Invitrogen, Waltham, MA). The slopes (calcein AM) or average fluorescence values (daunorubicin and pheophorbide A) per well were calculated and compared to the standard inhibitors 34−36. For compounds with an inhibitory power over 50% against a single target or 20% (+SEM) against all three ABC transporters, full-blown concentration-effect curves between 100 nM and 100 μM final compound concentrations have been generated. The data was processed using GraphPad Prism version 8.4.0, and IC 50 values were determined, applying three-or four-parameter logistic equations, whatever was statistically preferred.
Efficacy of Compounds 24 and 30 against ABCB1-expressing Cells. An MDR-reversal assay 2,55−59 was used to investigate the ability to reverse ABCB1-mediated MDR in A2780/ADR cells toward the cytotoxic ABCB1 substrate 33. For this purpose, 20 μL of the most potent ABCB1 inhibitors 24 or 30 (1 μM, 5 μM, 10 μM or 50 μM) was transferred onto clear 96-well flat-bottom plates and complemented with 160 μL of a cell suspension containing approximately 10,000 A2780/ADR cells. A dilution series of 33 at concentrations between 0.1 μM and 100 μM was generated, and 20 μL of each concentration was transferred onto the plates (final concentration: 0.01−10 μM). The plates were incubated for 72 h at 37°C and a 5% CO 2 -humidified atmosphere, before subsequent processing with the regular MTT assay as described above. The observed effects of compounds 24 and 30 were compared to the effect of 33 alone on A2780/ADR cells as well as its sensitive counterpart A2780 (∼10,000 cells/well).
Computational Analyses. Molecular Docking of Lead MCT1 Inhibitors. Molecular docking was conducted as described earlier 82−85 by applying Chimera version 1.10.2 with the Autodock vina 1.1.2 software in conjunction with the PyRx Virtual Screening Tool 0.8 and the Biovia Discovery studio. 86 The reference MCT1 inhibitors 7 and 8, as well as the most potent lead MCT1 inhibitors 24 and 30, were visualized by applying ChemDraw professional 17.1 and stored in mol file format. Energy minimization was accomplished with the Universal Force Field (UFF) within the PyRx. 87 For molecular docking of compounds 24 and 30, a recently released cryo-EM structure of MCT1 in complex with basigin-2 (PDB ID: 7CKR) 19 bound to the reference MCT1 inhibitor 7 was used, and the information deduced from its binding mode and binding affinity was used for subsequent analyses. A three-dimensional grid box (x = 67.52 Å; y = 62.25 Å; z = 66.43 Å) was adjusted, and the exhaustiveness parameter was set to 8.
Determination of Molecular−Structural and Physicochemical Properties. The numbers of H-bond donors, H-bond acceptors, and rotatable bonds as well as the physicochemical properties CLog P, MW, MR, and TPSA were calculated by applying the online web service SwissADME (http://www.swissadme.ch). 75 CLog P was determined by using the atomistic method, 76 while TPSA was determined using the fragment-based method. 77 ■ ASSOCIATED CONTENT * sı Supporting Information